Detection of chitinase activity by 2-aminobenzoic acid labeling of chito-oligosaccharides.
Chitinases are hydrolases capable of hydrolyzing the abundant natural polysaccharide chitin. Next to artificial fluorescent substrates, more physiological chito-oligomers are commonly used in chitinase assays. Analysis of chito-oligosaccharides products is generally accomplished by UV detection. However, the relatively poor sensitivity poses a serious limitation. Here we report on a novel, much more sensitive assay for the detection of chito-oligosaccharide reaction products released by chitinases, based on fluorescent detection, following chemical labeling by 2-aminobenzoic acid. Comparison with existing UV-based assays, shows that the novel assay offers the same advantages yet allows detection of chito-oligosaccharides in the low picomolar range.
Ghauharali-van der Vlugt
Karen
K
Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands.
Bussink
Anton P
AP
Groener
Johanna E M
JE
Boot
Rolf G
RG
Aerts
Johannes M F G
JM
D001681:Biological Assay; D002686:Chitin / Q000032:analysis / Q000737:chemistry*; D002688:Chitinases / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D009844:Oligosaccharides / Q000032:analysis / Q000737:chemistry*; D013379:Substrate Specificity; D062367:ortho-Aminobenzoates / Q000737:chemistry*
D009844:Oligosaccharides; D062367:ortho-Aminobenzoates; C031385:anthranilic acid; D002686:Chitin; D002688:Chitinases